tbuckley/cabot-search huggingface dataset:
Abstract P2033: Neddylation Regulates Cardiac Mitochondria Homeostasis
Authors: Jianqiu Zou, Wenjuan Wang, Juan Ayala, Masuko Ushio-Fukai, Tohru Fukai, Yisang Yoon, Weiqin Chen, Jie Li, Huabo Su
Journal: Circulation Research (2022)

Neddylation is a reversible post-translational modification that covalently attaches ubiquitin-like protein NEDD8 to target proteins via NEDD8-specific E1-E2-E3 enzymes. Through regulating the function of various cellular proteins, neddylation impacts diverse cellular processes and disease states. We have previously identified an indispensable role for neddylation in cardiac chamber maturation through sustaining YAP signaling in the developing heart. However, the importance of neddylation in post-mitotic hearts remains unclear. Here we report neddylation as a novel post-translation mechanism regulating mitochondrial homeostasis in adult heart. Inducible deletion of NAE1, a regulatory subunit of NEDD8 E1 enzyme, in adult mouse heart resulted in dilated cardiomyopathy, heart failure, and eventually premature lethality. The severe cardiac phenotype was preceded by accumulation of mitophagic vesicles and mitochondrial elongation, accompanied by pronounced upregulation of mitophagic markers and alterations in mitochondrial dynamics proteins. Moreover, we found that neddylation is required for mitochondrial ubiquitination and turnover. Consequently, pharmacological and genetic inhibition of neddylation accumulated damaged mitochondria, impaired mitochondrial membrane potential, inhibited mitochondrial respiration, and increased cardiomyocyte cell death in both neonatal and adult cardiomyocytes. Mechanistically, neddylation of Cullins is essential for the assembly of a functional multi-subunit Cullin-RING ubiquitin ligase, which appears to mediate mitochondrial ubiquitination following mitochondrial damage. Taken together, our findings suggest that neddylation serves as a novel mechanism surveilling mitochondrial quality and is essential for the function of normal adult heart.
Abstract P1131: Modeling Contractile Diseases Using Scalable 3D Engineered Heart Tissues For Drug Discovery
Authors: Nicholas Geisse, Bonnie Berry, Kevin Gray, Samir Kharoufeh, Shawn M Luttrell, Jesse Macadangdang, Christal Worthen
Journal: Circulation Research (2022)

Model systems that accurately recapitulate healthy and diseased function in a dish are critical for the development of novel therapeutics. For cardiac diseases, direct assessment of contractile output constitutes the most reliable metric with which to assess overall tissue function, as other ‘proxy’ measurements are poor predictors of muscle strength. 3D engineered muscle tissues (EMTs) derived from iPSCs hold great potential for modeling contractile function. Here, we have developed a platform and device that utilizes 3D EMTs in conjunction with a label-free magnetic sensing array. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source and is coupled with a highly parallel direct measurement of contractile strength. This approach enables the stratification of healthy and diseased phenotypes and facilitates compound safety and efficacy screening for evaluation of a drug’s effect on contractile output. We will present data from a drug (BMS-986094) that failed clinical trials due to unanticipated cardiotoxicity. We go on to show both the acute and chronic effects of doxorubicin in cardiac EMTs. Contractile force decreased in a dose dependent-like manner when the drug was applied continuously. Interestingly, a single 1uM bolus induced a transient effect that could be washed out over time. A repeat bolus, however, irreversibly abolished contraction, suggesting that repeat dosing may have a cardiotoxic effect on the muscle. These data demonstrate a first-and-only commercial platform for high-throughput assessment of 3D cardiac muscle contraction with potential for widespread adoption within the drug development field.
Abstract P1070: Role Of Vezf1 In Cardiomyocyte Development
Authors: Isaiah K Mensah, Hern Tan, Martin Emerson, Ming He, Humaira Gowher
Journal: Circulation Research (2022)

Cardiovascular diseases (CVDs) are the leading cause of high morbidity rates and death worldwide. The heart is the first organ that is formed during mammalian development. Progenitor cells that form the cardiovascular system (heart and blood vessels) originate from the mesoderm post gastrulation. To date, there remain considerable gaps in the molecular mechanisms that govern the differentiation of cardiac progenitor cells into a functional heart, knowledge of which will provide advancements in cardiac regenerative medicine. Vascular endothelial zinc finger 1 (Vezf1) is a nuclear protein expressed predominantly in the mesoderm of a developing mouse embryo. A recent study showed that diseased human myocardia had reduced expression of Vezf1 when compared to healthy hearts. However, the function of Vezf1 in heart development is largely uncharacterized and understudied. Using murine embryonic stem cells (mESCs), we sought to investigate the early role(s) of Vezf1 in cardiovascular development. We show that genetic ablation or conditional knockdown of Vezf1 in mESCs impairs cardiomyocyte differentiation, as determined by the absence of contractile activity in these mutants. The absence of cardiomyocytes from Vezf1 mutant cell lines was confirmed using gene expression analysis and immunofluorescence. We observed impaired mesoderm induction in Vezf1 mutants despite external stimulation of Wnt signaling using small molecule inhibitors, suggesting a potential role of Vezf1 in the modulation of genes in the signaling pathway. Indeed, our gene expression analysis and ChIP-qPCR indicate that Vezf1 potentially associates with the promoters of Wnt signaling genes to activate their expression. These studies point towards a model where Vezf1 regulates the formation of cardiac progenitor cells by modulating the Wnt signaling pathway. Our observations suggest that Vezf1 may sit at the center of determining the fate of mesodermal cells into cardiovascular progenitors, the absence of which impairs cardiomyocyte differentiation. This study is the first to delineate the functional role of Vezf1 in the early development of cardiomyocytes and may form the foundation for future cardiac regenerative medicine.
Abstract P1110: Identification Of Timp-1 As A Downstream Target Of The Coronary Artery Disease Risk Factor Adamts-7 In Atherosclerosis
Authors: M. Amin Sharifi, Michael Wierer, Jelena Milic, Aldo Moggio, Philipp Mueller, Julia Werner, Hendrik Sager, Juergen Bernhagen, Matthias Mann, Heribert Schunkert, Thorsten Kessler
Journal: Circulation Research (2022)

Background: The gene encoding the extracellular matrix (ECM) protease ADAMTS-7 was associated with coronary artery disease (CAD) in genome-wide association studies. ADAMTS-7 is expressed at all stages in human plaques and mice lacking Adamts-7 displayed reduced atherosclerotic plaque formation. While these findings render ADAMTS-7 a promising therapeutic target, the underlying mechanisms remain unknown. Methods and Results: Here, we sought to identify downstream mechanisms of ADAMTS-7 in atherosclerotic plaque formation. Targets of Adamts-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques in Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. The endogenous inhibitor of matrix metalloproteinases (MMP) Timp-1 was identified as a novel target of Adamts-7. Adamts-7 and Timp-1 were found to be co-localized in atherosclerotic plaques and co-immunoprecipitation (Co-IP) studies revealed TIMP-1 as the first putative target to bind to the catalytic domain of ADAMTS-7. In vitro degradation assays demonstrated that ADAMTS-7 degrades TIMP-1. Co-IP furthermore revealed less binding of TIMP-1 to its canonical target MMP-9 when ADAMTS-7 was present. In line, scaffolding and degradation of TIMP-1 by ADAMTS-7 impaired TIMP-1-mediated inhibition of MMP-9 in vitro. As a downstream mechanism, we investigated collagen content in atherosclerotic plaques of Apoe-/- and Apoe-/- Adamts7-/- mice after Western diet. Collagen stainings of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. Since ADAMTS-7 might exert its pro-atherogenic effects through interaction with TIMP-1, we established a TIMP-1-ADAMTS-7-interaction assay based on Förster resonance energy transfer (FRET) to identify inhibitors of this protein-protein interaction. Conclusion and Outlook: TIMP-1 represents a novel downstream target of ADAMTS-7 that can explain the role of this novel risk factor in CAD. Our FRET-based protein-protein interaction assay may be used for high-throughput screening to identifiy inhibitors and prevent the initiation or slow the progression of atherosclerosis.
Human bronchial epithelial cell dysfunction following in vitro exposure to nitrogen dioxide
Authors: JL Devalia, RJ Sapsford, DR Cundell, C Rusznak, AM Campbell, RJ Davies
Journal: European Respiratory Journal (1993)

Nitrogen dioxide (NO2), is a major air pollutant, that causes bronchoconstriction and bronchial hyperreactivity, and may also lead to damage and inflammation of the airway epithelium. We have cultured human bronchial epithelial cells and investigated the effect of exposure to NO2, for 20 min on epithelial cell membrane integrity and function in vitro. Epithelial cell membrane damage and permeability were assessed by release of 51Cr from prelabelled cells, and movement of 14C-labelled bovine serum albumin (BSA) across the bronchial epithelial cell monolayers. Ciliary beat frequency (CBF) of the cells was monitored by the analogue contrast enhancement technique, and arachidonic acid (AA) metabolism was investigated by analysis of radiolabelled AA metabolites generated from cultures prelabelled by incubation with [3H]-arachidonic acid. Exposure to 400 and 800 parts per billion (ppb) NO2 significantly increased the release of 51Cr from 0.9 +/- 0.4%, in control cultures exposed to 5% CO2 in air, to 9.7 +/- 3.2% and 13.9 +/- 3.5%, respectively. Similarly, NO2 also significantly increased the movement of 14C-BSA across the epithelial monolayers from 1.3 +/- 0.2%, in control cultures, to 2.7 +/- 0.2%, 3.8 +/- 0.4% and 5.1 +/- 0.5%, respectively, in cultures exposed to 100, 400 and 800 ppb NO2. Although NO2 attenuated the CBF of the cells at all concentrations investigated, this was significant only at the concentration of 2,000 ppb NO2.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract P3014: Dislocation Of Hexokinase1 From Mitochondria Results In Heart Failure With Preserved Ejection Fraction Through Hyper O-glcnacylation In Endothelial Cells.
Authors: Yuki Tatekoshi, Jason S Shapiro, Farnaz K Nejad, Mingyang Liu, Krithika Nayudu, Chunlei Chen, Hossein Ardehali
Journal: Circulation Research (2022)

Introduction: It is reported that endothelial cell (EC) dysfunction underlies the pathogenesis of heart failure with preserved ejection fraction (HFpEF). EC are known to highly rely on glycolysis to keep their function, but the metabolism of EC under the situation with HFpEF is poorly understood. Objective: We sought to elucidate the role of hexokinase 1 (HK1) in ECs for the development of HFpEF. Results: Isolated ECs from mouse hearts showed higher protein expression of HK1 than isolated cardiomyocytes and fibroblasts, suggesting HK1 has an important role in EC function. Immunogold-staining of HK1 in hearts from C57BL6 mice treated with high fat diet and LNAME, a mouse model of HFpEF, showed increased dislocation of HK1 from the mitochondria in ECs. To study the role of HK1 dislocation, we generated ΔE1HK1 mice with mitochondrial-binding domain of the endogenous HK1 replaced with Flag tag. Subcellular fractionation confirmed that HK1 was dislocated from mitochondria in these mice. Echocardiography showed that the mice developed impaired cardiac relaxation at 20 weeks of age and HFpEF at 40 weeks of age. Significant increased fibrosis and microvascular rarefaction (MR) were observed at 40 weeks of age, but only MR was observed at 20 weeks of age, suggesting EC dysfunction likely precedes and promotes the development of HFpEF in these mice. To study the angiogenic ability of ECs with HK1 dislocation, we isolated ECs from ΔE1HK1 hearts and performed a tubing assay, and observed significantly less tubing in ΔE1HK1 ECs. To elucidate the mechanism by which angiogenesis is reduced in ΔE1HK1 ECs, we next performed metabolomics analysis in these cells. Our data indicated that the levels of metabolites in hexosamine-biosynthetic pathway (HBP) were altered between wild-type (WT) and ΔE1HK1 EC. We next analyzed the levels of O-GlcNAcylation, and showed that ECs from ΔE1HK1 hearts have higher O-GlcNAcylation than those from WT. Finally, treatment with ST045849, an inhibitor of O-GlcNAc transferase (OGT), rescued the less angiogenic ability in ECs from ΔE1HK1 hearts. Conclusion: Our studies demonstrate that dislocation of HK1 plays an important role in the development of HFpEF through hyper-O-GlcNAcylation. Drugs that inhibit OGT may provide a therapeutic option for HFpEF.
Abstract P2067: Depleted Cardiac Energy Reservoirs And Type 2 Diabetes: The Unfulfilled Role Of Endogenous Creatine
Authors: Melanie Y White
Journal: Circulation Research (2022)

To maintain contractility, the heart turns over the cellular ATP pool six times per minute, with up to half replenished via the creatine:phosphocreatine system (Cr:PCr). This ‘safe’ energy reservoir arises from endogenous sources, through biosynthesis in the kidney and liver for delivery to the heart. Exogenous PCr drastically improves contractility following myocardial ischemia/reperfusion injury (I/R), as exemplified by cardioplegic PCr supplements provided during coronary artery bypass surgery. Considering clinical outcomes following I/R are typically worse in patients with Type 2 Diabetes (T2D), we hypothesized that T2D comorbidities that impact physiology of the kidney and liver may impair endogenous production of Cr and limit cardiac Cr:PCr. We employed a rat model that combined obesity and steatosis (high-fat diet), with pancreatic insufficiency (streptozotocin), to produce elevated blood glucose, serum triglycerides and reduced insulin sensitivity, consistent with a T2DM phenotype. Heart, liver, kidney and blood were sampled from T2D rats for histology and metabolomics by targeted mass spectrometry. Renal microanatomy showed little variation in response to T2D, while nuclear displacement and vacuolation was apparent in liver sections. We identified no change in renal Cr substrate guanidinoacetate (GAA) (0.7996±0.09; n -fold±SEM, p -value=0.524) or the serum GAA levels (0.95±0.13; n -fold±SEM, p -value=0.993). In the liver, GAA levels were depleted (0.468±0.04; p &lt;0.0001), with a comparable reduction of hepatic creatine abundance (0.391±0.07; p =0.003). Cardiac levels of both Cr and PCr were significantly depleted in the heart (Cr: 0.392±0.02, p &lt;0.0001; and PCr: 0.642±0.07, p =0.02). To confirm that this decrease in cardiac Cr:PCr did not simply reflect increased metabolism, we monitored cardiac creatinine and found only a modest decrease in T2D hearts (0.783±0.05; p =0.003), confirming a depletion in Cr bioavailability. In summary, hepatic derangement indicative of steatosis promoted impaired creatine biosynthesis to limit availability of Cr:PCr for restoration of ATP in the diabetic heart. In the absence of adequate ‘safe’ energy reservoirs, such hearts are compromised further following I/R injury.
Abstract P1058: Whole-cell Mechanical Loading And Unloading Triggers More Post-translational Modifications In α-actinin Than Myosin Activators And Inhibitors
Authors: Christopher Solis, Elisabeth DiNello, Chad M Warren, Kyle Dittloff, R John Solaro, Brenda Russell
Journal: Circulation Research (2022)

The hypothesis tested is that unloading of mechanical forces affects acetylation, ubiquitination, and phosphorylation of the actin-binding protein α-actinin located in the Z-disc. We applied targeted proteomics to interrogate the post-translational modification changes during loading and unloading. Cell morphology and post-translational modifications were determined in a mechanical intervention consisting of 1 Hz cyclic strain for 24 hr (loaded) followed by 6 hr rest (unloaded) of cultured neonatal rat ventricular myocytes (NRVMs). This was compared to a chemical intervention consisting of treating NRVMs with the myosin inhibitor Mavacamten (1 μM, 6hr) or activator Omecamtiv Mecarbil (0.5 μM, 6hr). Quantitative immunofluorescence showed both chemical and mechanical loading increased α-actinin content while Mavacamten decreased the α-actinin content. Mass spectrometry analysis of affinity-purified α-actinin revealed that the mechanical intervention led to increased levels of post-translational modifications compared with the chemical interventions. Specifically, α-actinin ubiquitination increased with mechanical loading-unloading; acetylation decreased with mechanical loading-unloading and increased with mechanical loading; and phosphorylation remained unchanged with mechanical loading-unloading but increased with mechanical loading. Fluorescence recovery after photobleaching (FRAP) experiments demonstrated that Mavacamten increased the dynamics of overexpressed YFP-tagged α-actinin and a GFP-tagged CapZ in NRVMs when compared to Omecamtiv Mecarbil treatments and controls. Overall, the results suggest a link between sarcomere homeostasis and mechanical forces via mechanisms involving acetylation, phosphorylation and ubiquitination of α-actinin and a second Z-disc protein, CapZ. These findings could have consequences for cardiac heart disease with abnormal sarcomeric proteostasis. Funded by NIH grants HL151825 (CS) and HL62426 (RJS, BR, and CMW).
Abstract P1085: Transient Receptor Potential Cation Channel Subfamily M Member 7 Deficiency Prevents Ventricular Tachycardia During Myocardial Ischemia Reperfusion
Authors: Xin Li, Jianlin Feng, Lixia Yue
Journal: Circulation Research (2022)

Introduction: Ventricular arrhythmia caused by myocardial ischemia reperfusion (myo-I/R) remain a substantial challenge worldwide. Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a Ca 2+ -permeable channel, and is involved in cardiogenesis and atrial fibrillation. However, the function of TRPM7 in ventricular arrhythmias during myo-I/R is unknown. Hypothesis: We propose that TRPM7 plays a role in ventricular arrhythmias during myo-I/R, therefore deletion of Trpm7 will protect mice against ventricular arrhythmia during myo-I/R. Methods: We used myo-I/R mouse model, in combination with patch-clamp, and various other techniques to investigate the potential role of TRPM7 in myo-I/R injury. Intracardiac programed electrical stimulation was performed to explore the susceptibility to ventricular arrhythmia, and in vitro assays were used for mechanism investigation. Results: We found that myo-I/R can readily induce spontaneous ventricular tachycardia (VT) in WT mice, whereas global Trpm7 deletion ( Trpm7 -/- ) dramatically reduced incidence of VT by 50.0% ( P &lt;0.05, n =10). In the program induced VT, Trpm7 -/- drastically shortened the duration of VT by 153.5 ± 24.0 (s) ( P &lt;0.05, n=10). As TRPM7 is highly expressed in both myocytes and cardiac fibroblast, we established myocyte-specific Trpm7 deletion and fibroblast-specific Trpm7 deletion mice. We found that TRPM7 in myocytes is responsible for VT induced by myo-I/R. To understand the mechanism by which TRPM7 is involved in VT, we evaluated electrphysiological properties of myocytes. The action potentials (AP) duration of ventricular myocytes isolated from WT mice, which significantly shortened after treatment of oxygen glucose deprivation, whereas Trpm7 -/- restored the AP by prolong the duration without influencing amplitude. Whole-cell patch clamp was performed to record L-type Ca 2+ and K + currents with sodium bisulfite treatment. Interestingly, Trpm7 -/- notably normalized the density of K + current back to the normal level when compared to WT myocytes subjected to oxidative stress. Conclusions: Our results indicate that TRPM7 in myocytes is involved in electrical remodeling during myo-I/R. Inhibition of TRPM7 channel function may serve as a potential target for myo-I/R induced ventricular arrhythmias.
Abstract P3099: Identification Of Hub Genes And Construction Of Protein-protein Interaction (PPI) Network Clusters Define Racially Distinct Signatures Of Ischemic Heart Failure
Authors: Sayan Bakshi, Luke Potter, Mark Pepin, Chae-myeong Ha, Adam R Wende
Journal: Circulation Research (2022)

Cardiovascular diseases, including ischemic heart failure (IHF), are the leading cause of mortality worldwide; furthermore, African Americans (AA) have 30% higher mortality than Caucasian Americans (CA). Our recent study correlates racial differences in socioeconomic status with DNA methylation (DNAm), altered gene expression, and mortality in HF independent of etiology. The current analysis focused on etiology-specific differences in DNAm with a specific focus on IHF, the related changes in gene expression, within self-reported race. RNA-seq expression ( P &lt; 0.05, ±1.5-fold) and array-based methylation ( P &lt; 0.05, ±5%) profiles were examined using cardiac biopsies from non-IHF (NIHF) and IHF AA (n = 9/5) and CA (n = 10/5) male patients. Network clusters were identified with MCODE (v2.0) and top hub genes identified via Cytohubba (v0.1). Both AA and CA patients showed similar numbers of IHF-specific differentially expressed genes (DEG) (591/365 AA; 439/486 CA; up/down). However, CA-specific methylation changes (29703/22703; up/down) were more abundant compared to AA (2904/2897; up/down). PPI network construction of these changes identified clusters unique to each group. The AA-specific upregulated cluster included AGPAT2, PPARG, and 10 other DEG associated with the fatty acid metabolism, while BMP2, SMAD6, and 4 other DEG that regulate BMP signaling and SMAD phosphorylation were downregulated. The CA-specific upregulated cluster included EZH2, CCNA1, and 18 other DEG that regulate mitotic sister chromatic separation, while EGFR, TNF, and 12 other DEG important for chronic inflammatory response formed the top downregulated cluster. Consistent with greater changes in DNAm the majority of DEGs in CA-specific clusters were also differentially methylated (58%) while fewer were in AA-specific clusters (17%). Within AA-specific PPI networks, PPARG/BMP2 (up/down) were the top ranked hub genes and KIF20A/KIT (up/down) in CA-specific networks. These racially distinct hub genes suggest altered regulatory mechanisms. Our analysis identified racially distinct clusters associated with specific pathways, which may connect the regulators of DNAm to gene expression and IHF, supporting the potential to use this information as prognostic biomarkers.